WO1998019523A1 - Model mouse for human diseases - Google Patents

Abstract

A model mouse for lipogenous type II diabetes and/or hepatic diseases accompanied by obesity, fatty liver or hyperlipemia, which is a novel congenic mouse created by introducing a variant ob (obese) gene (that of a type II diabetic mouse) into an inbred FLS mouse.

Description

 Specification

 Human disease model mouse Technical field

 The present invention relates to a disease model animal, and more particularly, to a novel condigenic strain mouse produced by introducing an ob (obese) gene having a mutation, for example, an ob gene of a type II diabetic mouse, into an inbred FLS mouse. And to a model mouse for obese type II diabetes and Z or liver disease.

Background art

 Diabetes in humans includes insulin-dependent diabetes mellitus (type I diabetes) caused by insulin secretion deficiency and absolute deficiency, as well as a decrease in the action of insulin in peripheral tissues and smooth uptake of glucose into tissues. Failure to do so is broadly divided into non-insulin-dependent diabetes mellitus (type II diabetes), which causes hyperglycemia. In the former case, Tengla Island is reduced in function or destroyed, resulting in insulin deficiency as seizures such as 脬 inflammation 免疫 autoimmune disease or severe type II diabetes. As a therapy, blood glucose levels are controlled by administration of insulin preparations. The latter is thought to have a diverse etiology, and its primary etiology has not yet been elucidated. Obesity, hyperinsulinemia, impaired glucose tolerance, hyperlipidemia, fatty liver, and the like have been reported as characteristics of the conditions that are commonly observed in human type II diabetes patients. Obesity in particular is a significant risk factor for developing type II diabetes. These conditions are not serious enough to be directly life-threatening, but can cause a variety of secondary complications, resulting in impaired patient function and longevity. It is a factor that leads to shortening of the period.

Not only in diabetes but also in research on the etiology and treatment of many human diseases, In recent years, disease model animals have played an important role. For example, NOD mice (Makino, S. et al .: Exp. Anim., 29, 1-13, 1980) and BB rats (Chappel, C.I. et al .: Metaboli sm) are used as models for type I diabetes. In particular, NOD mice play a very important role in elucidating the genetic factors of type I diabetes in humans.

 —On the other hand, the etiology and pathology of type II diabetes are so diverse that it is impossible to study the etiology and treatment of the disease using a single animal model. Therefore, there is a need to subdivide the characteristics of the various pathologies exhibited by human diabetes, develop animal models that are most suitable for each characteristic, and use them in research. At present, a large number of human type II diabetes model animals are provided, but existing model animals cannot cover all of the diverse pathologies of human type II diabetes. The creation of a model animal of type II diabetes is required for elucidating the etiology of human type II diabetes and conducting therapeutic research. In addition, with the increase in the prevalence of liver diseases including fatty liver and liver cancer associated with obesity and diabetes, the development of new animal models for spontaneous liver diseases is also desired.

Disclosure of the invention

 The present invention meets the above-mentioned demands, and is a mouse useful as a model animal for obese type II diabetes and / or liver disease, wherein an inbred FLS mouse is used as a background gene, and a normal ob gene product is produced. It is to provide a human disease model mouse not expressing.

Any genetic breeding technique used in the art can be used to produce the mouse mouse with a human disease of the present invention.For example, the ob (obese) gene having an existing mutation in the inbred FLS mouse Is preferably introduced by backcrossing. In addition, the mouse of the present invention is a molecular genetic technique, for example, FL It can also be produced by the knockout method of the ob gene of the S mouse. The knockout method is described in detail in Joyner, A. Shi. Ed .: Gene Targeting, IEL Press, 0xtord (1993).

 As the ob gene introduced by the above-described genetic breeding technique, an ob gene having a mutation derived from any mouse can be used as long as it is suitable for the purpose of the present invention. For example, single gene mutations that determine obesity traits in mice are known at at least six pairs of loci, but obesity due to the ob gene, one of which was already reported in 1950, Since then, C57BL / 6J-ob congenic mice, which mainly use C57BL / 6J as their genetic background, have been used as model animals for obesity and type II diabetes. The ob gene having this mutation was cloned in 1994 by Y. Zhang et al. (Y. Zhang et al., Nature 372 1994. 425-432), and the nucleotide sequence of its cDNA was also determined. Therefore, of the animals obtained in this manner, congenic animals homozygously containing the introduced ob gene do not express a normal ob gene product (lebutin) and are useful as the human type II diabetes model mouse of the present invention. is there.

 As a source of the ob gene to be introduced, a mouse having an ob gene having any existing mutation can be used provided that it meets the purpose of the present invention. / 6J-ob mice are the most common and are readily available.

 In the present specification, the ob gene introduced into or introduced into an inbred FLS mouse means an ob gene having a mutation.

Among the animals obtained in this way, with respect to the introduced ob gene, individuals with a heterozygous genotype (obZ +) represent the pathology of liver disease, and individuals with a genotype homo (ob / ob) Obesity, the origin of the introduced ob gene, obese type II diabetes model mouse (eg, C57BLZ6J-ob) 2 shows type diabetes and severe liver disease.

 As described above, the mouse of the present invention is a novel mouse having an ob gene introduced from an obese type II diabetic mouse using the FLS mouse as a background gene, and is homozygous (obZob) with respect to the ob gene. Includes individuals that are heterozygous (obZ +) with an individual. All of these individuals develop liver diseases such as fatty liver and liver tumors derived from FLS, but the former is further associated with obese type II diabetes. Both types of mice are useful as disease model mice, while the latter are also useful as parent animals for those disease model mice.

 Therefore, the present invention relates to a model mouse showing the pathology of obese type 2 diabetes and severe liver disease, wherein the inbred FLS mouse has a mouse having an ob gene having an existing mutation, such as C57BL / 6 The present invention provides a mouse (referred to as an ob-b-type mouse) which is produced by introducing the ob gene of a J-ob mouse by backcrossing and homozygously containing the introduced ob gene.

 The above ob / ob type mouse has, for example, the following characteristics.

 1) Strongly positive urinary glucose is observed in almost all cases in both sexes from 5 to 15 weeks of age;

 2) Both males and females have already gained about 5 to 10 g of body weight at 5 weeks of age from normal C57BLZ6 J mice;

 3) A remarkable hyperglycemic state of around 40 OmgZd 1 persists from the young age to 20 weeks of age;

 4) show hyperinsulinemia over 300 // U / m1;

 5) Abnormal glucose tolerance is observed;

6) Hyperlipidemia from young age to 20 weeks of age, blood cholesterol, triglyceride, and phospholipid concentrations each reach 40 OmgZd 1 or more; 7) Even under normal feed breeding, fatty liver mainly consisting of triglyceride deposition is exhibited from an early age, and liver weight is 5 to 10 times higher than that of normal mice; and 8) Liver tumors occur spontaneously in elderly individuals .

 Furthermore, the present invention is a disease model animal showing a pathological condition of liver disease and a mouse useful for producing a fertile type II diabetes model mouse, wherein the inbred FLS mouse has an ob gene having a mutation. A mouse having the ob gene, such as an ob / + mouse, which is produced by backcrossing the ob gene having the mutation of a C57BL / 6J-ob mouse. Is what you do.

 The above ob / + mouse has, for example, the following characteristics.

 1) Fatty liver mainly due to triglyceride deposition develops at an early age under normal feed breeding; and

 2) Liver tumors spontaneously occur in elderly individuals.

 Thus, obZ + mice are useful as model mice for liver diseases, particularly liver tumors, and are also useful as parent animals in the production of the above ob / ob model mice. That is, the obZob type mouse can be produced by crossing obZ + mice and selecting an individual having the ob gene homozygously.

BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing the change in the appearance rate of urinary sugar in FLS-obZob mice and C57BLZ6 J-ob / ob mice.

 FIG. 2 is a graph showing the results of an oral glucose tolerance test in female FLS-obZob mice and C57BLZ6J-obZob mice at the age of 10 weeks.

FIG. 3 is a schematic diagram showing the process of producing FLS-ob / ob mice. FIG. 4 is a diagram showing an outline of the production of subtracted cDNA using a PCR-select cDNA subtraction kit (CLONTECH).

 FIG. 5 is a mimetic diagram of a photograph showing expression of OPN (osteopontin) and PAP (pancreatitis-associated protein) mRNA in FLS-obZob mouse liver tissue analyzed by Northern blot. 6M represents 6-month-old liver tissue, 12M represents 12-month-old liver tissue, and 18M represents 18-month-old liver tissue (liver tumor). One actin is a control.

 FIG. 6 shows the nucleotide sequence of PAP cDNA derived from liver tumor tissue of FLS-obno ob mouse mouse. The upper row shows the sequence of FLS-ob / ob mouse PAP, and the lower row shows the sequence of mouse PAP.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, an example of the method for producing the disease model mouse of the present invention, the properties thereof, and an example of the method of breeding will be described in detail.

The mouse of the present invention is based on the FLS (Fatty Liver Shionogi) mouse (Yoshio Kishimoto et al .: Proceedings of the 31st Annual Meeting of the Japanese Society of Experimental Animal Science, PP119, 1984), which is characterized by spontaneous development of fatty liver and liver tumors. As a gene, a mouse obtained by deleting the expression of the normal ob gene product [levbutin protein (obesity-suppressing hormone)] by introducing an ob gene derived from a type II diabetic mouse by gene breeding techniques. In addition, in addition to the pathological condition of the liver disease derived from the FLS gene, individuals homozygous for the introduced ob gene (obZob-type mice) show severe pathology of type II diabetes, and are extremely model animals of the disease. Useful. This ob / ob-type mouse is a useful model for obesity type II diabetes in humans and the pathophysiology and pathogenesis of complications such as fatty liver and hyperlipidemia. Animal. It is also useful as a model animal for spontaneous liver tumors to study the pathological change of liver cancer from liver damage including fatty liver in humans. On the other hand, as described later, an individual having an introduced ob gene in a heterozygous state (ob + type mouse) does not show the pathology of type II diabetes, but expresses pathologies such as fatty liver and liver tumor. Like the above ob / ob type mice, it is useful as a model animal for spontaneous liver tumors for studying the pathological changes of liver cancer from liver damage including fatty liver. Further, this ob / + mouse is extremely important as a parent animal of the above-mentioned type II diabetes model mouse (obZob).

 Therefore, the phrase “disease model mouse” or “mouse of the present invention” used in the context of the present invention is usually used in a type II diabetes model mouse (Homozygous for the introduced ob gene). obZob type mouse), but may also include a mouse having the ob gene heterozygously (obZ + type mouse). The meaning used is clear from the context.

 The disease model mouse of the present invention can be obtained from the above-mentioned FLS mouse by a genetic breeding technique. That is, it is a conduit strain produced by introducing the ob gene of, for example, an existing type II diabetic mouse into the FLS mouse by backcrossing and named FLS-ob. As described below, the obtained animal exhibits characteristics that combine the characteristics of both the FLS mouse and the mouse that is the source of the ob gene.

 For the purpose of the present invention, as described above, a mouse having an ob gene having a mutation includes C57BL / 6J-, which is characterized by obesity, appearance of urinary glucose, hyperglycemia, hyperinsulinemia, and insulin resistance. Ob mice (Ingalls, AM et al .: J. Hered, 41, 317-318, 1950) are preferred, but not limited thereto.

The FLS-ob strain of the present invention obtained by introducing the ob gene of the C57BLZ6J-ob mouse into the FLS mouse is characterized by the following characteristics of both the FLS mouse and the C57BL / 6J-ob mouse. It has the following characteristics. The following specific explanation is based on the combination Related to animals.

 (1) The ob gene introduced into the mouse of the present invention is a simple recessive gene located on chromosome 6 on an autosomal chromosome (Bray, GA et al .: Physiol. Rev., 59, 719-809, 1979). It has the following genetic features:

 a) Due to simple recessive inheritance, the obesity trait presumed to be crossed with this gene is expressed only in individuals homozygous for the ob gene.

 b) Since these genes are on autosomes, these traits are expressed in both sexes.

 c) As a result of positional cloning by Zhang et al., C57BLZ6

The ob gene of the J-ob mouse has a nonsense mutation in its sequence and cannot produce a normal ob gene product (Nature, 372, 425-431, 1994). That is, even in the mouse of the present invention, a normal ob gene product is not produced in an individual having the ob gene homozygously.

 d) The ob gene is expressed in adipose tissue (Zhang et al., 1994).

 e) The product, or protein, encoded by the normal ob gene, named leptin, has been shown to be an obesity-suppressing hormone (Halaas, JL et al .: Science, 269, 543-546, 1995).

 f) The mouse strain of the present invention has the same background gene because the ob genotype is maintained by crossing heterozygous (obZ +) parents, but the ob genotype of the individual is obZo Separates into ob / + or + ノ +. The characteristics of the FLS-ob / ob mouse of the present invention, which is related to the above-mentioned genetic predisposition and has the ob gene homologously, correspond to the corresponding C57BL / 6J-obZob mouse, and a similar purpose already existing. As shown below, compared with the model mouse.

(2) In the mouse of the present invention, at the age of 5 weeks, almost all males and females were found to be strongly positive for urinary glucose. This condition continues until about 15 weeks of age. After that, the incidence of urinary sugar decreased Initially, it becomes about 50% at the age of 35 weeks, and almost no urinary sugar is observed after 60 weeks of age. These observations were made using urine glucose test paper under non-fasting conditions. C 57 B LZ6 J—The incidence of urinary glucose in obZob mice

In males, about 40% of males and about 15% of females were reported even at the time of the test (Mashiko Nishimura: Glycaturia, 15, 426-430, 1972). As a result of observation by the present inventors under the same breeding conditions as the mouse of the present invention, the appearance rate of urinary glucose in C57BLZ6J-obZob mice was approximately 45% for males and approximately 20% for females during peaks. The appearance of urine sugar after 40 weeks of age was hardly observed.

 Therefore, it was confirmed that the diabetes of the present invention persisted for a longer period of time and the incidence of diabetes was higher than that of the C57BL / 6J-obZob mouse (see FIG. 1). ).

雌が26. 2 g(n=19)を示す。 30週齢頃から体重増加は停止し、 雌雄とも約 55〜 65gとなる。 一方、 C 57 BL/6 J一 obZobマウスの 5週齢における 体重は雄が平均 17. 4g(n=l 1)、 雌が 16. 7g(n= 15 )であり、 本 発明マウスは C 57BL/6 J—obノ obに比べて若齢時の体重増加が顕著 である（第 1表参照）。 (3) The mouse of the present invention exhibits obesity presumed to be caused by the introduced ob gene. Males average weight at age 5 weeks 26.

Females show 26.2 g (n = 19). At about 30 weeks of age, weight gain stops, and both males and females weigh about 55 to 65 g. On the other hand, the average body weight of C57BL / 6J-one obZob mice at the age of 5 weeks was 17.4 g (n = l 1) for males and 16.7 g (n = 15) for females. The weight gain at the young age is remarkable compared to / 6 J-obno ob (see Table 1).

Table 1

 Average body of FLS-ob / ob mouse and C57IU76J-ob / ob mouse at each age! : (G)

 FLS-ob / ob mouse

In addition, with aging, both mice strains begin to lose weight around the age of 50 weeks. C57BLZ6 J—ob / ob mice reported to weigh up to 9 Og (Bates, M. ff. Et al .: Am. J. Physiol., 180, 301-303, 19). 55), but the values may vary between facilities and colonies. C As a result of breeding the FLS-obZob mouse of the present invention under the same conditions, C57BL / 6J- Ob The maximum weight of ob mice is about 55 to 65 g, and no difference was observed between the mice and the mice of the present invention.

C57BL / 6 J—As a genetically obese diabetic animal other than ob / ob mice, C57BL / Ks J -db / db (Hummel, KP et al .: Science, 153, 1127-1128, 1966) And KK (Kyoji Kondo et al .: experimental animals, 6, 107-112, 1957) and KK- ^Ay (Nishimura, M: Exp. Animal, 18, 147-157, 1969). In addition, C3H mice (Fenton, PF et al.

J. Nutr., 49, 319-331, 1953) is known to cause diet-induced obesity. In the rat, Zucker fatty (Zucker, L. M. et al .: J. Hered.,

52, 275-273, 1961), Wistar fatty acid (Ikeda, H. et al .: Diabetes, 30, 1045-1050, 1981), 0 L E T F (awano, K. et al .: Diabetes, 41,

1422-1428, 1989). In the case of mice alone, C57 BLZ6 J—obZob mice and C57 B LZKs J—dbZdb mice began to gain weight relatively early, with peak weights at 6 months and 3 months, respectively, Herberg et al. (Metabolism, 26, 59-99, 1977) and Thur lby et al. (Br. J. Nutr., 39, 397-402, 1978), both of which are classified as juvenile obesity. In contrast, KK mice and KK- ^Ay mice gradually develop obesity as they age, and can be called adult-type obesity (Nakamura, M. et al .: Proc. Japan Acad, 38, 348-352,

1962).

As described above, since the weight of the mouse of the present invention is significantly higher than that of the C57BL / 6 J-ob / ob mouse at the age of 5 weeks in both males and females, juvenile obesity progresses very rapidly. Positioned as a model. (4) The mouse of the present invention already exhibits hyperglycemia at the age of 5 weeks, and the blood glucose concentration at this time is 400 to 45 OmgZdl under non-fasting conditions in both sexes. Such marked hyperglycemia is observed for up to about 20 weeks. Blood glucose levels subsequently declined, reaching 200-250 mg / dl at 40 weeks of age.

 — On the other hand, C57 BLZ6 J— The blood sugar level of obZob mice is 250-3 OOmg / dl at 5 weeks of age, and shows high blood sugar, but its degree is mild. Thereafter, the blood glucose level increases with age and reaches a peak of about 40 OmgZdl (about 10 weeks of age). However, the blood glucose level of C57BL / 6J-obZob mice falls earlier than that of this mouse. It drops to 200-25 OmgZdl at 20 weeks of age.

 Thus, the mouse of the present invention maintains a hyperglycemic state for a longer period of time as compared with the C57BLZ6 J-obZob mouse.

 (5) Like the C57BLZ6J-ob / ob mouse, the mouse of the present invention exhibits hyperinsulinemia which is one of the characteristics of type II diabetes.

 It has been reported that the blood insulin level of C57BL-6J-obZob mice shows a 10- to 50-fold higher value than that of normal mice (Genuth, S .: Endocrinology, 88, 1230-1238, 1971). The blood insulin concentration of C57BL / 6J-ob / ob mice maintained by the present inventors is 350-450 / ζυη1. This is about 10 times that of normal mice (C57BL6J mice).

 The blood insulin concentration of the mouse of the present invention also shows 300 to 450 AiU ml, and its level is not significantly different from that of the C57BL / 6 J-ob_ / ob mouse.

Among the existing type II diabetes model mice, C57BL-6J—ob_ob mice do not show very high hyperglycemia, but are classified into a group that causes marked hyperinsulinemia. BL / 6 J-dbZdb mice (Kaku, K. et al .: Diabetologia, 32, 636-643, 1989) also belong to this group. The mice of the present invention also had almost the same level of blood as C57BLZ6 J-ob / ob mice. Sugars and hyperinsulinemia can be classified in this group

 In addition, C57BLKsJ-dbZdb mice exhibit intense hyperglycemia, and blood glucose levels reach 70 OmgZdl, but insulin is not as high as C57BLZ6J-obZob mice (Herberg, L. et ah, 1977). The blood glucose level of KK mice is lower than that of C57BLZ6J-ob ob mice (30 Omg / dl), while insulin shows 1,200 zU / ml (Nakamura,. Et al., 1962).

 As a result of a glucose tolerance test using female mice of the present invention, marked abnormalities in glucose tolerance were observed at 10 weeks of age. The initial blood glucose level after an overnight fast showed 213 ± 13 mgZdl (n = 5) and increased to 564 ± 23 mgZdl after 30 minutes of oral glucose tolerance (2 gZKg body weight). In addition, the blood glucose level after 180 minutes of loading was 393 ± 13 mg / dl, indicating a delayed recovery of the blood glucose level. In contrast, C57BL / 6J-obZob female mice of the same age had an initial blood glucose level of 128 ± 18 ragZdl (n = 5), and 5 13 ± 17 mg / dl 30 minutes after glucose loading. After that, blood glucose dropped relatively quickly, and recovered to 154 ± 1 Orag / dl after 180 minutes (see Fig. 2).

 From these results, it is clear that the mice of the present invention show hyperglycemia and hyperinsulinemia, which are characteristics of type II diabetes, and show more severe abnormal glucose tolerance than C57B LZ6 J-obZob mice. .

 (6) The mouse of the present invention exhibits severe fatty liver from a young age, which has the following characteristics.

 a) Remarkable liver enlargement and yellow to whitening are observed as gross findings. b) Liver wet weight at the age of 15 weeks exceeds 5 g for both males and females, and increases with aging, with some individuals exceeding 10 g.

At 15 weeks of age, the above values were for normal mice, C57BLZ6J mice. 948

5-6 times, equivalent to about 2 times that of C57BLZ6 J-obZob mice. That is, the degree of liver hypertrophy associated with fatty liver is more severe in the mouse of the present invention than in C57BL / 6J-ob / ob.

 c) Light microscopic observation of the liver formalin-fixed section reveals diffuse oil red staining positive lipid droplets in the hepatocytes of the mouse of the present invention.

 d) The average value of cholesterol per 1 g of liver of the mouse of the present invention at 15 weeks of age was 15. Omg for both males and females. The triglyceride amount was 89.lmg for males and 98.2 nig for females. 28.6, females show 32.2 mg.

Compared to C57BL / 6J-ob mice, the amount of triglyceride is about 1.5 to 1.7 times higher and that of phospholipid is 1.4 to 1.5 times higher, but the amount of cholesterol There is no difference (see Table 2).

Table 2

 FLS-ob / ob mouse and C57BL / 6J ob / ob mouse

 Liver lipid content at 15 weeks of age (mg / g liver)

FLS-ob / ob mouse

 TG; triglyceride, CH0; cholesterol,

 PL; phospholipid, *; significantly different from FLS-ob / ob mice (p <0.05).

 The above results support that this mouse has fatty liver mainly due to triglyceride deposition.

e) Glutamate oxaloacetate transaminase (GOT) activity and glutamate pyruvate transaminase activity (GPT), which are indicators of liver damage, were 950 IU / U 980 Ιϋ / l for both males and females in the 15-week-old mouse of the present invention. These results indicate severe liver dysfunction. None of the existing hereditary obese diabetic animals exhibit such severe liver damage, and the development of severe fatty liver is a major feature of the mouse of the present invention.

 (7) The mouse of the present invention exhibits remarkable hypertriglyceridemia, which is not observed in the C57BL / 6J-obZob mouse. From the results of measurement on females under non-fasting conditions, the blood triglyceride concentration of the mouse of the present invention at the age of 5 weeks was 314 ± 32 mgZdl (n = 5), which is a normal mouse C57BL / 6 Approximately 8 times that of J mice, 2 times that of FL S mice that develop fatty liver

 It was 71 ± 1 Omg / "dl (n = 5) in C57BL / 6J-ob / ob mice of the same age, and the mice of the present invention showed about 4-fold higher values.

ピークとして減少し、 20週齢で 432 ± 51 ngZdl (η = 5)、 40週齢で 187±4 lmgZdl(n=5)となった。 The blood triglyceride level of the mouse of the present invention was 634 ± 5 at 10 weeks of age.

It decreased as a peak, reaching 432 ± 51 ngZdl (η = 5) at 20 weeks of age and 187 ± 4 lmgZdl (n = 5) at 40 weeks of age.

On the other hand, in C57BLZ6 J-ob / ob mice, the blood triglyceride level varied between about 60 and 9 OmgZdl from the age of 5 weeks to 40 weeks. Menahan et al. (Metabolism, 32, 172-178, 1983) reported that C57B L / 6 J-obZob mice showed low blood triglyceride levels despite obesity. Salmon et al. (Biochem. J., 136, 551-563, 1973) and Elliot et al. (Biochem. Biophys.) show that obZob mice show higher levels of triglyceride secretion from the liver into the blood than normal mice. Acta., 343, 30 7-323, 1974), Yen et al. (Biochem. Biophys. Acta., 441, 213-220, 1976), Kaplan et al. (Am. J. Physiol., 240, E101-E107, 1981). However, because of the high Lipoprotein lipase activity in adipose tissue and the like, which promotes triglyceride catabolism in blood, apparently Gasquet et al. (Biochem. J., 127, 445-447, 1972) and Matsushita (Clinical Science, 11, 866-873, 1975) have reported low ceride values.

The Zucker Fatty rat, a hereditary obese diabetic rat, exhibits hyperlipidemia because the release of triglyceride from the liver surpasses that in spite of the elevated triglyceride turnover in the blood. However, although its value is about twice as high as that of normal rats, it is about 15 Omg / dl (Takerao to, S. et al .: Horm. Metab. Res., 7, 242-246, 1975).

 On the other hand, in the case of the mouse of the present invention, blood cholesterol and phospholipid concentrations also show high values. The peak is observed around 10 weeks of age, and shows about 450 mg / dl (n = 5) and about 730 mg / dl (n = 5), respectively, under non-fasting conditions.

 On the other hand, in C57BL / 6J-obZob mice, it is about 25 Omg / dl (n = 5) and about 330 mg dl (n = 5), respectively. These conditions persist in both mice until about 20 weeks of age.

 From the above, it can be seen that the mice of the present invention show more severe hyperlipidemia than C57BL / 6J-obZob mice including not only triglycerides but also cholesterol / phospholipids.

 It is known that diabetes is often accompanied by hyperlipidemia. Hyperlipidemia is a serious risk factor for cardiovascular diseases such as arteriosclerosis, and as cardiovascular diseases are rapidly increasing as a cause of diabetes, it is important to understand the causes of these diseases. It is. However, many of the current obese diabetic model animals do not share diabetic conditions such as hyperinsulinemia and hyperglycemia, and abnormal lipid metabolism such as obesity and hyperlipidemia. It is considered to be highly useful as a type II diabetes model animal with obesity and hyperlipidemia.

(8) C57BL / 6 J-obZob mice die after 50 weeks of age under free-feeding conditions Austin et al. (Experimental

Gerontology, 19, 121-132, 1984).

 Comparing the survival rates of the C57BL / 6J-I obZob mouse maintained by the present inventors and the present inventors, in the case of the mice of the present invention, both males and females began to die at about 65 weeks of age. Both males and females had a survival rate of 50% at 76-78 weeks of age.

 On the other hand, the 50% survival rate of C57BLZ6 J-obZob mice is 83 weeks old for males and 90 weeks old for females, and the survival days of the mice of the present invention tend to be short. Examination of the autopsy findings at the time of death of the mouse of the present invention and the C57BL / 6J-obZob mouse revealed that in the mouse of the present invention, 43 of 43 males and females (100%) showed yellowing of the liver, Nodule formation was observed in 18 patients (42%). In C57BLZ6J-obZob mice, liver yellowing appeared in 100% of individuals, but nodule formation remained in 2% (50 cases of both sexes were searched).

 In the mice of the present invention, hydronephrosis / kidney abscess was observed in 23% of the individuals, whereas in C57BL / 6J-obZob mice, it was 6.

When the mouse of the present invention becomes old, a tumor naturally occurs in the liver. FLS mice have similar characteristics. Spontaneous liver tumors are rare in rats, and the spontaneous incidence of hepatocellular carcinoma in F344 rats used for carcinogenesis studies is goodman et al. (Toxicol. Appl. Pharmacol., 48, 237-248, 1979), Haseman et al. Natl. Cancer Int., 75, 975-984, 985) and Maekawa et al. (Mutagens and toxicity, 14, 22-41, 1981). % Is reported. It has been found that spontaneous liver tumors are relatively more common in mice than in rats. It has been reported that the incidence of C 3 H strains is 72-99% for males and 58-96% for females (Akihiko Maekawa et al., 1981). In the mouse of the present invention, nodule formation and hematoma frequently occur in the liver from about 8 months of age. Light microscopy shows that these are hepatocellular tumors and vascular tumors. Hepatocellular carcinoma is grossly recognized as neoplastic nodules of various sizes. Histologically, it compresses the surrounding tissues and shows trabecular or solid growth, with some tubule-like structures. Vascular tumors are visibly dark red, cystic, and contain large amounts of blood. Histologically, it is recognized as hemangiomas or hemangioendothelioma. The frequency of these tumors increases with age.

 Human liver cancer generally develops from chronic inflammation (hepatitis) through cirrhosis. Tissue necrosis caused by inflammation causes liver fibrosis and compensates for increased hepatocyte regeneration increases the risk of mutation (Nobuhiro Sato et al .: New Trends in Molecular Medicine (Japan Society for Clinical Metabolism), P116-120, Nakayama Bookstore, Tokyo, 1996). In humans, the causes of hepatitis and liver cancer are predominantly viral (Saito, I. et al .: Proc. Natl. Acad. Sci. USA, 87, 6547-6549, 1990). On the other hand, chronic fatty liver is also considered to cause hepatitis (Schaffner, F. et al .: Prog. Liver Dis., 8, 283-298, 1986). However, it is not yet clear whether inflammation due to hepatic steatosis can be a risk of carcinogenesis. Therefore, the mouse of the present invention, which involves chronic severe fatty liver and liver cancer, is promising as a model for examining the relationship between steatohepatitis and carcinogenesis.

Since the cirrhotic phase of humans is not histologically similar to that of humans, it is necessary to clarify how the liver tumors occurring in this mouse match the tumor biological characteristics of human liver cancer. is there. This is also important from the viewpoint of the model animals being exposed to human diseases. In order to examine the characteristics of the liver tumor of this mouse from a molecular biology perspective, the inventors subtracted a gene expressed in tumor tissue. (Hara, E. et al .: Nucleic Acids Ees., 19, 7097-7104, 1991)

 The subtraction method is one of the methods for identifying genes specifically expressed in various cell types. That is, when identifying a gene that is expressed in A cells but not in B cells, a labeled cDNA probe is prepared from mRNA of A cells and a hybridization reaction is performed with mRNA or cDNA of B cells. . As a result, genes expressed in both cells have a double-stranded structure, but genes expressed only in A cells remain single-stranded because they have no partner to react. Then, this single-stranded cDNA is recovered and the gene is identified (Michael Kriegler: Genetic Engineering of Animal JIS, P168-200, Takara Shuzo, Kyoto, 1994). In this experimental system, oligo- (dT) -cellulose was used to extract from liver tumor tissue of 18-month-old FLS-ob / ob mice and from 2-month-old FLS-ob / ob liver tissue. -A subtracted cDNA was obtained according to the protocol of select cDNA subtraction kit (CLONTECH). Figure 4 shows an outline of the method for preparing the subtracted cDNA. This was ligated to a TA cloning vector (Invitrogen) and transformed into Escherichia coli to prepare a subtracted cDNA library. A cDNA fragment that was found to be expressed in tumor tissue was screened using a plasmid dot blot, and the nucleotide sequence was determined using a DNA sequencer (Model 307: Applied Biosystems). EMBL / GenBank / DDBJ, which is a gene database, was used for homology search.

As a result, in the liver tumor tissue of the mouse of the present invention, the expression of osteobontin (0 PN) mRNA RNA associated protein (PAP) mRNA was observed. When the expression of both nRNAs with aging was analyzed in a Northern blot, the expression of PAP mRNA was found only in the 18-month-old tumor tissue. On the other hand, OPN mRNA was already expressed at 6 months of age, and its expression tended to increase with aging (Fig. 5). In FIG. 5, 6M represents 6-month-old liver tissue, 12M represents 12-month-old liver tissue, and 18M represents 18-month-old liver tissue (liver tumor). — Actin is a control.

 0 1 ^ has recently become a ligand for 044 and has been suggested to be involved in cancer invasion and metastasis (Weber, GF et al .: Science, 271, 509-512, 1996). Lectin expressed in the knee during the recovery from inflammation is thought to be involved in the regeneration and proliferation of Teng cells (Keim, V. et al .: Digestion, 29, 242-249, 1984). The PAP gene has been used in rat (Iovanna, J. et al .: J. Biol. Chera., 266, 24664-24669, 1991) and human (Orelle, B. et al .: J. Clin. Invest). , 90, 2284-2291. 1992) and mice (Itoh, H. et al .: Biochim. Biophys. Acta, 1172, 184-186, 1993). PAP is expressed only in the small intestine in normal individuals (Itoh, H. et al., 1993). In 1992, Lasserre et al. Found a gene expressed in human primary liver cancer and named it HIP (Cancer Res., 53, 5089-5095, 1992). HIP was expressed in 25% of patients with primary liver cancer. In the same year, this HIP was found to be identical to human PAP and is thought to be involved in tumor cell growth (Lasserre, C. et al., 1992).

 The present inventors performed cloning of the PAP cDNA derived from the liver tumor of the mouse of the present invention, and compared the sequence with that reported previously (Itoh, H. et al., 1993). However, the untranslated regions were found to be different (Fig. 6). In FIG. 6, the upper row shows the sequence of FLS-ob / ob mouse PAP, and the lower row shows the sequence of mouse PAP (Itoh, H. et al., 1993). SEQ ID NO: 1 shows the nucleotide sequence of PAP cDNA derived from the liver tumor of the mouse of the present invention.

No expression of PAP in animal tumors has been reported so far, and human liver cancer The fact that such tumor biological characteristics were confirmed in the liver tumor of the mouse of the present invention strongly suggests the usefulness of the mouse as a liver cancer model.

 Based on the characteristics described above, the mice of the present invention showed no clear difference in the degree of obesity (maximum body weight) compared to the existing obese type II diabetic mice C57BLZ6 J-obZob, but obesity progressed earlier. It is characteristic in the appearance of urinary sugar for a long time and the persistence of hyperglycemia. In addition, they exhibit severe fatty liver mainly due to triglyceride deposition, and cause hyperlipidemia not seen in C57BL / 6 J-ob / ob mice. These indicate that the mice of the present invention have good clinical findings that are frequently observed in human type II diabetic patients, and in that respect they have superior properties to C57BL / 6J-ob / ob mice. .

 Further, as described above, since the etiology of type II diabetes has not been elucidated, it is considered that the pathological condition of the mouse of the present invention is also useful for exploring the etiology. Furthermore, in the mouse of the present invention, since liver tumors occur spontaneously at high frequency, it is considered to be useful as a model animal for studying the mechanism of liver tumor development and examining the relationship between abnormalities in lipid metabolism and liver tumor development. .

 Hereinafter, the present invention will be described with reference to Examples, but these do not limit the present invention.

Example 1 Animal production process and breeding method

(1) Animal origin

 FLSCFatty Liver S hionogi) Mice: (Yoshio Kishimoto et al .: Proceedings of the 31st Annual Meeting of the Society of Experimental Animal Science, PP119, 1984)

A strain of inbred strain produced from outbred dd mice that was introduced to Laboratories by Shionogi & Co., Ltd. in 1955 from the Central Research Institute for Experimental Animals, and used for histological and biochemical observations. It has been shown that fatty liver develops on the basis of this, and liver hypertrophy and yellowing frequently occur. At the end of 1995, 119 generations TJP97 / 03948

It is established as an inbred strain.

 C57B LZ6 J—obZob mouse: (Ingalls, A. M. et al .: J. Hered, 41, 317-318, 1950):

 A strain obtained by introducing a gene controlling obesity traits (ob gene, Ingalls et al., Supra) into C57BL / 6J mice. The number of animal generations at the time of acquisition by the present inventors was 21 generations, but by the end of 1995, the animals had been passaged for 10 more generations (31 generations in total).

(2) Animal creation

 The congenic mice of the present invention were created by a known back-cross method. “Backcross” means a cross between the first hybrid and either parent used for the first cross. In this case, the parent does not have to be the parent individual used in the original cross (first hybrid), but it must belong to the same line and have the same genotype (edited by Fujiwara Kosaku et al.) : Encyclopedia of Laboratory Animal Science, PP427-428, Asakura Shoten (Tokyo), 1989)). The specific method for producing the animal of the present invention is as follows.

 1) A female FLS mouse and a male C57BL / 6 J-ob mouse were bred to introduce an ob gene into the FLS mouse as a background gene to obtain the first cross (N1). In the first generation, individuals with ob locus ob / + and individuals with + / + segregate 1: 1. From this, the ob / + individuals with the ob gene are crossed with FLS mice.The obesity trait is expressed only in individuals having the ob gene in the homozygous (ob / ob). Neither individuals possessing terrorism (ob +) nor individuals possessing wild-type gene (+ / +) are obese and cannot be distinguished in appearance. Hetero individuals were identified by progeny test mating, which was performed by re-crossing in offspring.

Offspring born from heterozygous crosses (Nl F 1) have ob ob genotype ob / +: ob / ob: Separates to + / + (2: 1: 1). In other words, if there is an individual who exhibits obesity, the parent can be said to be a heterosexual individual in both sexes. Obese individuals can be distinguished from their weight at about 3 weeks of age.

 2) Among the parents that gave birth to the obese individuals, males were further bred to female FLS mice, and backcrossed in the same manner as described above. Backcrossing was performed over a total of 12 generations (N12), and the rate of replacement of genes other than the ob gene in the mouse strain of the present invention was 99.

Estimated at 97%. A schematic diagram of the backcrossing is shown in FIG.

 3) Backcrossing From the 12th generation, the offspring (N12F1) born by crossing between ob / + are designated as the F1 generation, and siblings are crossed thereafter. The number of generations reaches 13 generations.

 At the time of filing the present application, the number of inbred generations has not yet been reached, but since the two strains used for the production of the congenic animal of the present invention are both inbred, the genetic characteristics of the mouse of the present invention have already been fixed. Seems to be. Inbreeding has continued. The mouse strain of the present invention was named FL S-ob mouse.

 (3) mating style

 Since individuals with the ob gene homozygous (obZob) lack fertility, both males and females breed using individuals with the ob gene heterozygous (obZ +). It is unclear whether the lack of fertility is congenital or secondary. As described above, the offspring obtained by crossing heterozygotes are separated into three ob genotypes, obZob, ob / +, and + Z +, and the separation rates are 1: 2: 1, respectively. Confirmation of heterozygotes used for mating is also performed by progeny test mating as described above. Since April 1995, we have been using the ob-gene determination method based on PCR-RFLP to select for heterozygous individuals to be used as parent animals.

 (4) Reproductive performance

The pregnancy rate of the mouse of the present invention is 89.0% (pregnancy confirmed in 281 mated female parents) The number of C57BLZ6 J-ob mice was 80.2% higher than that of C57BLZ6 J-ob mice (239 out of 298 breeding females, of which 239 were confirmed to be pregnant). The average number of offspring was 5.7 for the mouse of the present invention (250 female parents gave birth to 1,425), and 4.6 for C57BL6J-ob mice (239 females gave birth). 1.

095 births). The weaning rate was 87.2% for the mouse of the present invention (1,224 weaning out of 1,403 parents) and 89.1% for C57BL6J-ob mouse (892 out of 1,001 parents). Weaning), and the weaning rate of the mice of the present invention tended to be somewhat lower. In addition, among the pups born from the reproductive performance so far, the acquisition rate of obZob mice was 17.3% for the mice of the present invention, and the average number of obZob mice acquired per female parent was 0.9. . In the case of C57 BLZ6 J-ob mice, the obZob mouse acquisition rate was 15.8%, and the average number of ob / ob mice acquired per female parent was 0.6.

(5) Animal breeding method

1) Rearing environment

Since the mice of the present invention are all SPF (specific pathogen free; non-specific pathogen free), they are bred in a barrier environment. The environmental conditions in the breeding room are set at room temperature 23 ± 1 ° C, humidity 60 ± 5%, and light / dark switching cycle (illuminance 30 Olux) every 12 hours. The ventilation frequency in the breeding room is set to 15 times per hour, and fresh outside air is introduced through a hepar filter. The inside of the barrier environment is kept at a positive pressure from the outside to prevent foreign matter from entering. The breeders should thoroughly disinfect their face and hands and wear sterilized harp garment, shoes, hood, mask and gloves before entering the room. Items will be delivered after autoclaving, ethylene oxide gas sterilization, UV sterilization, or disinfectant treatment. In addition, the breeding facility conducts a microbial search once a month, and the animals are HVJ., MH V. ヽ Bordetella bronchiseptica ^ Corynebacterium Kutscheri, Hyco. plasma pulmonis ^ Tyzzer 's disease ^ Salmonella typhimurium _N Salmone 11a sp., Staphylococcus aureus ヽ Pseudomonas aeruginosa ^ Pasteurel la pneumotropica. .

 2) Feed and drinking water

 The mouse of the present invention is fed with a breeding solid feed C A-1 manufactured by CLEA Japan as an animal feed after autoclaving (121 ° C, 7 minutes). Drinking water is supplied by an automatic water supply device after passing tap water through a filter with a pore size of 3 m and a running water sterilizer (UV sterilization).

 3) Animal properties

 The mice of the present invention are easy to handle in the order of their properties, and they rarely fight even if several males and females are kept in the same cage.

 4) Notes on rearing

 These mice are prone to heavy drinking and urination during the onset of diabetes. Therefore, the floor covering (white flakes, etc.) tends to become dirty, so it is necessary to replace it as appropriate and keep the cage floor clean. When using water bottles, attention should be paid to the lack of drinking water. C57BLZZJ-obZob mice are known to be hypothermic (Kaplan,. L. et al .: Am. J. Physiol., 227, 912-915, 1974). From this, it is considered that this mouse is also the same, and it is considered that plastic cages are more preferable for breeding than metal cages in terms of heat retention.

 5) About distribution

The non-insulin-dependent diabetic mouse with obesity, fatty liver and hyperlipidemia of the present invention can be distributed from this institution. Sequence listing

 SEQ ID NO: 1

 Array Length: 7 5 6

 Sequence type: nucleic acid

 Number of chains: double strand

 Topology: linear

 Sequence type: cDNA to mRNA Organism name: FLS-oVob mouse

 Tissue type: Liver tumor

Array

 ACACCATCCA GATCTCTGGA AGACAGACAA GATGCTGCCT CCAACAGCCT GCTCCGTCAT 60 GTCCTGGATG CTGCTCTCCT GCCTGATGCT CTTATCTCAG GTTCAAGGTG AAGACTCCCT 120 GAAGAATATA CCCTCCGCAC GCATTAGTTG CCCCAAGGGC TCCCAGGCTT ATGGCTCCTA 180 CTGCTATGCC TTGTTTCAGA TACCACAGAC CTGGTTTGAT GCAGAACTGG CCTGCCAAAA 240 GAGGCCTGGA GGACACCTCG TATCTGTGCT CAATAGCGCT GAGGCTTCAT TCTTGTCCTC 300 CATGGTGAAG AGAACAGGAA ACAGCTACCA ATACACTTGG ATTGGGCTCC ATGACCCCAC 360 TCTGGGTGCA GAACCCAATG GCGGTGGATG GGAATGGAGT AACAATGACG TGATGAATTA 420 CTTTAACTGG GAGAGGAACC CATCTACTGC CTTAGACCGT GCTTTCTGTG GCAGCTTGTC 480 AAGAGCTTCT GGATTTCTAA AATGGAGAGA TATGACATGT GAGGTGAAGT TGCCCTATGT 540 CTGCAAATH ACTGGTTAAA CTTATCAGAC AGCAAACATC CCGAATTTGT CTTGAAGAGC 600 ATCATGGACA AGGGACAA TGTGAAGACT CACQAGAAA AAGCATTTTC TATCTACAGT 660 CCACATTAGA GCCTTAATCT GCTCHTCCA TATCTGTCTT TAGTCCTTTT GGTATAAGTT 720 TGGGCTCAAT TCTAAAATAA AAATAAGCTT TCTGTC 756

Claims

The scope of the claims  1. An inbred FLS mouse as a background gene and a human disease model mouse in which a normal ob gene product is not expressed.  2. A human disease model mouse, which is a congenic animal obtained by backcrossing the ob gene having a mouse-derived mutation into an inbred FLS mouse.  3. The model mouse according to claim 1 or 2, wherein an ob gene derived from a C57BLZ6J1 ob mouse, which is a model mouse for obese type II diabetes, is introduced.  4. The model mouse according to claim 2 or 3, wherein the introduced ob gene has a homologous or a hepatic mouth.  5. The model mouse according to claim 2, wherein the introduced ob¾ gene is homozygous.  6. The model mouse according to any one of claims 1 to 5, having the following characteristics: 1) Strongly positive urinary glucose is observed in almost all cases in both sexes from 5 to 15 weeks of age;  2) Both males and females show significant weight gain from young age;  3) remarkable hyperglycemic state persists from young age to 20 weeks of age;  4) show hyperinsulinemia;  5) Abnormal glucose tolerance is observed;  6) Hyperlipidemia due to elevated blood cholesterol, triglyceride, and phospholipid concentrations from young to 20 weeks of age;  7) Severe fatty liver mainly due to triglyceride deposition develops at an early age under normal feed breeding; and  8) Liver tumors spontaneously occur in aged individuals. 7. Indicating the pathology of obese type II diabetes and severe liver disease, any of claims:! To 6 The model mouse according to 1.  8. The model mouse according to any one of claims 2 to 4, which has the introduced ob gene in heterologous form.  9. The model mouse according to any one of claims 1 to 4 and 8, having the following characteristics: 1) Under normal diet, develop fatty liver mainly from triglyceride deposition from an early age; and  2) Liver tumors spontaneously occur in elderly individuals.  10. The model mouse according to claim 8 or 9, which shows a pathological condition of liver disease.  11. A human disease model showing the pathology of obese type II diabetes and severe liver disease, comprising breeding the mouse according to any one of claims 8 to 10 and selecting an animal having the ob gene homozygously. Mouse manufacturing method.